... Reliable restriction enzymes satisfy all your research needs /p...Restriction endonucleases are used in many molecular biology protocols...Prior to assaying samples for contaminants the unit activity of each ...

Restriction endonucleases are used in many molecular biology protocols,
making the functional purity of critical importance. Each restriction enzyme
preparation must be free of contaminants that can potentially interfere with
either DNA cleavage or subsequent reactions that use digested DNA (i.e.,
ligation or sequencing reactions). Such contaminants might include other
site-specific endonucleases that produce unwanted DNA fragments, exonucleases
that destroy the integrity of the cut site by cleaving nucleotides from the
resultant 5 or 3
termini of the fragments, and phosphatases that inhibit ligation reactions by
removing the 5 phosphate from DNA ends. To ensure
that Stratagenes enzymes consistently meet the needs of the research
community, we impose strict quality control procedures in analyzing every enzyme
preparation. In most instances, the testing conditions are designed to be more
rigorous than typical laboratory applications. In this article, we describe the
assays used to establish the functional purity of restriction endonuclease
preparations.

Unit Activity Determination

Prior to assaying samples for contaminants, the unit activity of each enzyme
sample is accurately determined. One unit of activity is defined as the amount
of enzyme required to completely digest 1 g of substrate DNA in 1 hour at the
appropriate temperature under the optimal assay conditions. This information is
provided on the Certificate of Analysis provided with each restriction
endonuclease. Restriction enzyme activity is substrate-dependent; therefore,
when digesting a new substrate, we recommend titrating the amount of enzyme
added. The unit activity of a repr
esentative sample from each packaging of a
restriction endonuclease is confirmed prior to shipment to demonstrate that no
loss of enzymatic activity occurs upon packaging.

Overdigestion of Substrates

When enzymes are tested under standard digestion conditions (1 U of enzyme/g
substrate DNA), trace levels of contaminating DNase activities might not be
detected. To more stringently assess the functional purity of each enzyme
preparation, Stratagene incubates a 4- to 100-fold excess of enzyme with
substrate DNA. After a 16-hour incubation at the appropriate temperature, the
banding pattern from these overdigested samples is compared to the
signature-banding pattern produced by the enzyme in 1 hour. If the restriction
endonuclease preparation is free from contaminating endonuclease and exonuclease
activities, the overdigested reactions will exhibit the identical banding
pattern characteristic of the 1-hour reactions. The maximum number of units that
clearly exhibit this pattern in the overdigestion reactions is reported on the
Certificate of Analysis provided with each enzyme. This overdigestion assay is
also employed on a representative sample following aliquoting and packaging to
confirm that contaminating DNase activity is not introduced during packaging.

Detecting Exonuclease Contaminants

The integrity of a restriction enzyme cut site can be destroyed by the
presence of exonucleases that can digest single-or double-stranded ends.
Stratagene specifically assays for exonuclease activity by incubating an excess
of restriction endonuclease for 4 hours with a mixture of single- and
double-stranded, [3H]-thymidine-labeled genomic E. coli DNA.
Contaminating exonuclease is indicated if over 0.6% of the 3H-labeled
DNA added to the reactions is detected as trichloroacetic acid-soluble counts.

Ligation Assay

Figure
1
p>

Stratagene confirms the functional purity of each restriction enzyme
preparation using an assay that simulates a typical cloning protocol.
In this assay, substrate DNA is completely digested using a three-fold
excess of enzyme, and the enzyme is removed using StrataClean
resin. The digestion products are ligated using Stratagenes T4 DNA
ligase and then recut with the same restriction enzyme. Samples of the
cut, ligated, and recut DNA are subsequently analyzed by agarose gel electrophoresis
(Figure
1). If the ends of the digested DNA have been compromised by contaminating
exonucleases or phosphatases, the recognition site will no longer be intact,
and the cut product will not ligate efficiently or, when recut, yield
a banding pattern identical to the initial digest. Stratagene reports
the percent of ligated and recut substrates on the Certificate of Analysis.

Nicking Assay

Figure
2

To analyze for contaminating endonuclease or topoisomerase activities,
Stratagene incubates an excess of restriction endonuclease with supercoiled
plasmid DNA that lacks the restriction enzyme recognition sequence. Following
a 4-hour incubation at the appropriate temperature, the conversion of
supercoiled DNA (RF I) to the open circular form (RF II) is monitored
by agarose gel electrophoresis (Figure
2). This conversion is indicative of a single nick in the RF I DNA.
If greater levels of contamination are present, linear DNA (RF III) might
also be observed. Stratagene guarantees that its restriction endonuclease
preparations will generate <5% conversion to RF II DNA and no detectable
conversion to RF III DNA.

Blue-White Cloning Assay

Figure
3

Another highly sensitive assay that models the use of restriction enzymes
is the blue-white color selection assay. In this assay, Stratagenes
pBluescript II SK phagemid vector (Figure
3), which contains the lacZ reporter gene, is cut at a single
position within the multiple cloning site using a five-fold excess of
enzyme. A phenol-chloroform extraction is used to purify the linearized
vector. The vector is ligated and transformed into Epicurian Coli
XL1-Blue competent cells, which are then plated on a medium containing
X-gal and IPTG. Functional b-galactosidase
(determined by blue colonies) is produced only for those transformations
with plasmids containing uninterrupted reading frames. If the integrity
of the DNA ends produced during the digestion reaction is compromised
(i.e., a nucleotide is deleted), the lacZ gene is interrupted,
and white colonies result. Enzymes tested in this assay (those commonly
used in cloning applications) must produce less than 5% white colonies.

Stability Testing

The unit activity of each restriction endonuclease is confirmed at least once
every six months. This stability testing guarantees that enzymes are fully
active when delivered to your laboratory. Most Stratagene restriction enzymes
are guaranteed to be stable for 18 months from the date of testing.

Conclusions

Stratagene recognizes that high-quality reagents are imperative for molecular
biology applications and, therefore, commits to providing an extensive line of
rigorously qualified restriction enzymes. We stringently establish the
functional purity of every enzyme preparation to ensure optimal performance.

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